(1) Field of the Invention
This invention relates generally to the regulation of apoptosis and to polypeptides, including both antagonists and agonists, which regulate apoptosis as well as to polynucleotides encoding these polypeptides, and, more particularly, to the novel death agonist, BID and the polynucleotide encoding BID.
(2) Description of the Related Art
Programmed cell death, referred to as apoptosis, plays an indispensable role in the development and maintenance of homeostasis within all multicellular organisms (Raff, Nature 356: 397-400, 1992 which is incorporated by reference). Genetic and molecular analysis from nematodes to humans has indicated that the apoptotic pathway of cellular suicide is highly conserved (Hengartner and Horvitz, Cell 76: 1107-1114, 1994 which is incorporated by reference). In addition to being essential for normal development and maintenance, apoptosis is important in the defense against viral infection and in preventing the emergence of cancer.
Considerable progress has been made in identifying the molecules that regulate the apoptotic pathway at each level. Of note, both positive and negative regulators, often encoded within the same family of proteins, characterize the extracellular, cell surface and intracellular steps (Oltvai and Korsmeyer, Cell 79: 189-192, 1994 which is incorporated by reference).
One such family of proteins that constitutes an intracellular checkpoint of apoptosis is the BCL-2 family of proteins. The founding member of this family is the apoptosis-inhibiting protein encoded by the bcl-2 protooncogene which was initially isolated from a follicular lymphoma (Bakhshi et al., Cell 41: 889-906, 1985; Tsujimoto et al, Science 229: 1390-1393, 1985; Cleary and Sklar, Proc Natl Acad Sci USA 82: 7439-7443, 1985 which are incorporated by reference). The BCL-2 protein is a 25 kD, integral membrane protein of the mitochondria. This factor extends survival in many different cell types by inhibiting apoptosis elicited by a variety of death-inducing stimuli (Korsmeyer, Blood 80: 879-886, 1992 which is incorporated by reference).
The family of BCL-2-related proteins has been defined by sequence homology that is largely based upon conserved motifs termed bcl-homology domains. (Yin et al, Nature 369: 321-323, 1994 which is incorporated by reference). Bcl-homology domains 1 and 2 (BH1 and BH2) domains have been shown to be important in dimerization and in modulating apoptosis (Yin et al., Nature 369: 321-323, 1994 which is incorporated by reference). A third homology region, BH3, has also been identified as important to dimerization as well as apoptosis (Boyd et al., Oncogene 11: 1921-1928; Chittenden et al., Embo J 14: 5589-5596, 1995 which are incorporated by reference) as has been a fourth homology region, BH4, near the amino terminal end of some family members (Farrow and Brown, Curr Opin Genet Dev 6: 45-49, 1996 which is incorporated by reference)(see FIGS. 13A-13E).
Members of this family can heterodimerize and, in most cases, homodimerize as well. The ratio of death antagonists (BCL-2, BCL-X.sub.L, MCL-1 and A1) to agonists (BAX, BAK, BCL-X.sub.S and BAD) determines which homodimers or heterodimers are formed and the balance of these is believed to determine whether a cell will respond to an apoptotic signal (Oltvai and Korsmeyer, Cell 79: 189-192, 1994 which is incorporated by reference). Thus, dimerization between agonists and antagonists is competitive. For example, the death promoting molecule BAX forms homodimers that favor death whereas BAX will also form heterodimers with BCL-2 or BCL-X.sub.L (Oltvai et al., Cell 74: 609-619, 1993 which is incorporated by reference) and the formation of these heterodimers results in inhibition of cell death. Mutagenesis studies have revealed that intact BH1 and BH2 domains of the antagonists (BCL-2, BCL-X.sub.L) are required for them to heterodimerize with BAX and to repress cell death (Yin et al., Nature 369: 321-323, 1994; Sedlak et al. 1995 which are incorporated by reference). Conversely, deletion analysis has indicated that the BH3 domain of death agonists (BAK, BAX) is required for them to heterodimerize with BCL-X.sub.L or BCL-2 and to promote cell death (Chittenden et al., Embo J 14: 5589-5596, 1995; Zha et al. 1996 which are incorporated by reference). However, other mutations in BCL-X.sub.L have been noted to disrupt heterodimerization with BAX, but retain death repressor activity (Cheng et al., Nature 379: 554-556, 1996 which is incorporated by reference). This suggests that these molecules might also work independent of one another. Recently, the first X-ray and multidimensional NMR structure of a family member, BCL-X.sub.L, was determined (Muchmore et al., Nature 381: 335-341, 1996 which is incorporated by reference). It was found that .alpha. helices correspond to the BH1-BH4 domains and that a hydrophobic pocket results from the close spatial proximity of the BH1, BH2 and BH3 domains.
The BH3 domain may play a role in the promotion of death by some of the death agonists although in others such as BAD the BH3 domain is not present. An important role for the BH3 domain has been suggested for death agonist family members that lack both the BH1 and BH2 domains, but have a BH3 domain. One such family member is BCL-X.sub.S. This protein, which is translated from an alternatively spliced version of the mRNA encoding BCL-X.sub.L, inhibits the ability of BCL-2 protein to enhance the survival of growth-factor deprived cells (Boise et al. Cell 74: 597-608, 1993 which is incorporated by reference). BCL-X.sub.S also contains a BH4 homology region as does BCL-X.sub.L.
BIK is another death agonists having a BH3 but not BH1 or BH2 domains and this protein also lacks a BH4 domain (Boyd et al., Oncogene 11: 1921-1928, 1995 which is incorporated by reference). Like the classic family members such as BCL-2 itself, this protein has a C-terminal hydrophobic domain which appears to function as a signal/anchor segment to enable transmembrane localization (Nguyen et al., J Biol Chem 268: 25265-25268, 1993 which is incorporated by reference).
The BH3 domain of BAK, an agonist with BH1, BH2 BH3 and C-terminal membrane localization domains, has been postulated to be of central importance in mediating the cell death promoting effect of this family member. This conclusion was based upon deletion studies which identified the BH3 region as necessary for induction of cell-death and upon the retention of cell killing activity by a 50 amino acid polypeptide fragment including BH3 but excluding BH1 and BH2 which indicated that the BH3 domain is sufficient for eliciting cell death.
Some disease conditions are believed to be related to the development of a defective down-regulation of apoptosis in the affected cells. For example, neoplasias may result, at least in part, from an apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signals. Furthermore, some DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitize the host cellular machinery to drive their own replication and at the same time modulate apoptosis to repress cell death and allow the target cell to reproduce the virus. Moreover, certain disease conditions such as lymphoproliferative conditions, cancer including drug resistant cancer, arthritis, inflammation, autoimmune diseases and the like may result from a down regulation of cell death regulation. In such disease conditions it would be desirable to promote apoptotic mechanisms and one advantageous approach might involve treatment with a cell death agonists having a BH3 domain which has been identified as being an important agonist determinant.
Furthermore, in certain disease conditions it would be desirable to inhibit apoptosis such as in the treatment of immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, wound-healing, and the like. In the treatment of such diseases it would be desirable to diminish the cell death agonist activity of endogenous proteins containing BH3 domains. Thus it would be desirable to identify new members of the BCL-2 family which have cell-death agonist properties by virtue of the presence of a BH3 domain and to utilize these as a basis for treatment modalities in advantageously modulating the apoptotic process in disease conditions involving either inappropriate repression or inappropriate enhancement of cell death.